Page 106 - Tyrosine-Based Bioconjugations - Jorick Bruins
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Chapter 6
Figure 4. SDS-PAGE analysis of optimization of SPOCQ on Tras-Y1 and Tras-Y2. Both antibodies were run without reagents (Lane A, F), with only mTyr (Lane B, G) and with varying protein and BCN-lis concentrations (Lanes C-E and H-J).
A final attempt to perform SPOCQ on Tras-Y1 and Tras-Y2 was based on performing the reaction in phosphate buffer instead of PBS, as sodium chloride is known to inhibit mTyr.22 An overnight reaction at 4 °C with 22 mg/mL was performed and analyzed with HPLC (SI S6.6). No significant improvement on yields were detected, however even more changes were detected to the heavy chain signals of both antibodies. The signals were shifted significantly and the intensity became lower than that of the light chain, this while the antibody was only stored for 28 days at 4 ˚C.
An overview of the HPLCs of the antibodies without the presence of mTyr or BCN-bearing compounds was made (Figure 5), and it depicts clearly the rapid change of the antibodies. It is unknown what is happening exactly, but Grünewald et. al. did report on the antibody losing
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